Simulation of Enzyme-Substrate Encounter with Gated Active SitesRebecca C. Wade, Brock A. Luty, Eugene Demchuk, Jeffry D. Madura, Malcolm E. Davis, James M. Briggs and J. Andrew McCammonNature Structural Biology, Vol. 1, No. 1, pp. 65-69 (1994) [PubMed 7656010]We describe a brownian dynamics simulation method that allows
investigation of the effects of receptor flexibility on ligand binding
rates. The method is applied to the encounter of substrate,
glyceraldehyde 3-phosphate, with triose phosphate isomerase, a
diffusion-controlled enzyme with flexible peptide loops at its active
sites. The simulations show that while the electrostatic field
surrounding the enzyme steers the substrate into its active sites, the
flexible loops appear to have little influence on the substrate binding
rate. The dynamics of the loops may therefore have been optimized during
evolution to minimize their interference with the substrate's access to
the active sites. The calculated and experimental rate constants are in
good agreement.
Molecular Modeling Methods. Basic Techniques and Challenging ProblemsBogdan Lesyng and J. Andrew McCammonPharmacology & Therapeutics, Vol. 60, Issue 2, pp. 149-167 (1994) [PubMed 7912833]An overview is presented of computer modeling and simulation methods
that play an increasing role in drug design: quantum chemical methods,
molecular mechanics, molecular dynamics and Brownian dynamics. The
application of molecular dynamics for the prediction of thermodynamic
properties like free energy differences and binding constants is
discussed. The Brownian dynamics method is presented in connection with
the calculation of effective electrostatic forces using the
Poisson-Boltzmann equation, which allows one to sample ligand-binding
geometries and to predict the kinetics of diffusion-limited enzyme
reactions. New techniques that have recently been extensively developed,
such as the global energy minimization and quantum-classical dynamics
methods, are also introduced. The molecular modeling methods are
illustrated with selected examples.
Open "Back Door" in a Molecular Dynamics Simulation of AcetylcholinesteraseM.K. Gilson, T.P. Straatsma, J.A. McCammon, D.R. Ripoll, C.H. Faerman, P.H. Axelsen, I. Silman and J.L. SussmanScience, Vol. 263, Issue 5151, pp. 1276-1278 (1994) [PubMed 8122110]The enzyme acetylcholinesterase generates a strong electrostatic field
that can attract the cationic substrate acetylcholine to the active
site. However, the long and narrow active site gorge seems inconsistent
with the enzyme's high catalytic rate. A molecular dynamics simulation
of acetylcholinesterase in water reveals the transient opening of a
short channel, large enough to pass a water molecule, through a thin
wall of the active site near tryptophan-84. This simulation suggests
that substrate, products, or solvent could move through this "back
door," in addition to the entrance revealed by the crystallographic
structure. Electrostatic calculations show a strong field at the back
door, oriented to attract the substrate and the reaction product choline
and to repel the other reaction product, acetate. Analysis of the open
back door conformation suggests a mutation that could seal the back door
and thus test the hypothesis that thermal motion of this enzyme may open
multiple routes of access to its active site.
Prediction of pH-Dependent Properties of ProteinsJan Antosiewicz, J. Andrew McCammon and Michael K. GilsonJournal of Molecular Biology, Vol. 238, Issue 3, pp. 415-436 (1994) [PubMed 8176733]We describe what may be the most accurate approach currently available
for the calculation of the p
Kas of ionizable groups
in proteins. The accuracy is assessed by comparison of computed
p
Kas with 60 measured p
Kas in a
total of seven proteins. The overall root-mean-square error is 0.89
p
Ka units. Linear regression analysis of computed
versus measured p
Kas yields a slope of 0.95,
y-intercept of -0.02 and a correlation coefficient of 0.96. The proposed
approach also picks out many of the shifted p
Kas of
groups in enzyme active sites and special salt bridges. However, it does
yield several over-shifted p
Kas and tends to
underestimate p
Ka shifts which result from
desolvation effects. We examine the ability of the new approach to
reproduce the dependence of protein stability upon Ph, using the
ionization polynomial formalism. Overall features of the stability
curves are reproduced, but the quantitative agreement is not
particularly good. The reasons for the disagreement may have to do both
with insufficient accuracy in the theory and with uncertainty in the
nature of the unfolded state of proteins. The methodology described here
is based upon finite difference solutions of the Poisson-Boltzmann
equation. Its success depend upon the use of the rather high protein
dielectric constant of 20. However, theoretical considerations and the
fact that p
Ka shifts which result from desolvation
are underestimated here imply that the dielectric constant of the
protein interior actually is lower than 20. We suggest that the high
protein dielectric constant improves the overall agreement with
experiment because it accounts approximately for phenomena which tend to
mitigate p
Ka shifts and which are not specifically
included in the model. These include conformational relaxation and
specific ion-binding. Future models based upon a low protein dielectric
constant and treating such phenomena explicitly might yield improved
agreement with experiment.
Combined Conformational Search and Finite-Difference Poisson-Boltzmann Approach for Flexible Docking. Application to an Operator Mutation in the Lambda Repressor-Operator ComplexM. Zacharias, B.A. Luty, M.E. Davis and J.A. McCammonJournal of Molecular Biology, Vol. 238, Issue 3, pp. 455-465 (1994) [PubMed 8176736]The N-terminal domain of the phage λ repressor binds as a dimer
to its palindromic DNA operator sequence. In addition to a
helix-turn-helix DNA recognition motif, the first six amino acids of the
phage λ repressor form a flexible peptide segment which wraps
around DNA. Site-directed mutagenesis studies have shown that amino acid
replacements or partial removal of the arm structure, or changes in the
DNA sequence contacting the N-terminal arm, can lower the
repressor-operator binding affinity by several orders of magnitude. The
finite-difference Poisson-Boltzmann approach in combination with a
conformational search procedure was used to study energetic
contributions of the λ arm to repressor-operator recognition
based on the high resolution X-ray structure. It allows for the local
relaxation of the structure upon changing the DNA sequence in the
λ arm binding region. A simplified potential energy function
including torsional, truncated Lennard-Jones and approximate
electrostatic terms is used in the initial step to screen out
energetically unfavorable structures. The electrostatic energy of
selected conformations is subsequently calculated more accurately using
the finite-difference Poisson-Boltzmann approach. The method was applied
to study the effect of a C→T mutation at position 6 of the
consensus half-site of the operator. This base-pairs contacts Lys4 which
is part of the arm segment. Keeping only the Lys4 side-chain mobile and
with the wild-type DNA operator sequence, several conformations close to
the X-ray structure were identified as those with lowest energy. In the
case of the DNA mutation, lowest energy conformations differed
significantly from those selected for the wild-type sequence. These
initial calculations indicate that the approach might be a useful tool
to estimate conformational and energetic effects upon mutagenesis of
protein-DNA complexes.